Phytophthora resistance gene of catharanthus and its use

ABSTRACT

The present invention relates to a disease resistant Catharanthus seed, Catharanthus plant, Catharanthus variety and Catharanthus hybrid. More specifically, the invention relates to a Catharanthus plant having resistance to the fungal disease Phytophthorao. The invention also relates to an increased level of resistance to aphids and other pests and an increased level of total alkaloid content. The invention further relates to the method of crossing Catharanthus plants containing the resistance to Phytophthora to produce disease resistance and increased total alkaloid level in Catharanthus plants and in other crop plants.

BACKGROUND OF THE INVENTION

The present invention relates to a Catharanthus seed, a Catharanthusplant, a Catharanthus variety and a Catharanthus hybrid which contain alevel of resistance to the disease Phytophthora parasitica. The presentinvention also relates to a Catharanthus plant product, an increasedlevel of alkaloid content, and an increased resistance to certaininsects.

Catharanthus roseus (L.) G. Don, also called periwinkle, or vinca,originates from Madagascar and belongs to the family of the Apocynaceae.This species is frequently grown annually from seed or cuttings intemperate climates for use in summer bedding or as a pot plant for theconservatory or the windowsill. C. roseus has long been grown as anornamental in tropical regions of the world. As a consequence of itsself-seediness (ability to pollinate itself and readily form matureselfed seed) it is now widely naturalized in many tropical regions.Commonly known as Madagascar periwinkle or vinca (not to be confusedwith the separate genus Vinca) this ornamental is commonly propagatedfrom seed and less frequently from stem cuttings.

Periwinkle is valued for its bushy habit, large desirable flowers,tolerance to heat, drought and direct sunlight. In addition to itshorticultural merits, C. roseus contains alkaloids used to retardcertain diseases, such as leukemia.

Catharanthus species are well known for their production of indolealkaloids as described in Farnsworth, Lloydia 24:105-139 (1961);Sevestre-Rigouzzo, et al., Euphytica 66:151-1569 (1993). Catharanthusroseus is one of the most thoroughly investigated of plants with regardto its constituent indole alkaloids, of which more than 70 have beenisolated from the whole plant as described in Balsevich and Hogge, J.Nat. Prod., 51: 1173-1177 (1988). Alkaloids are generally known ascompounds biologically active against pathogens and herbivores asdiscussed in vanDam, et at., Oecologia 95:425-430 (1993). Indolealkaloids are potent antifeedants. Tested in a bio-assay using a 0.04%solution, vinblastine and catharanthine appeared to be the mostdeterrent alkaloids against the polyphagous Spodoptera larvae asdescribed in Meisner, et al., J. Econ. Entomol. 74:131-135 (1981);Chockalingam, et at., J. Environ. Biol. 10:303-307 (1989). Periwinkleextracts have also been shown to have strong inhibitory activity againstseveral bacterial genera. Farnsworth Lloydia 24:105-139 ( 1961) alsodescribed the anthelmintic activity of alkaloid fractions of C. roseus.Alkaloids obtained from C. roseus provide the basis of a 22-year oldindustry yielding well over a hundred million dollars annually asdescribed in Raven, Diversity 9:49-51 (1993). The anticancer activity ofvinblastine and vincristine, both isolated from C. roseus, is welldocumented in the pharmaceutical industry.

To date there has been no known resistance to Phytophthora inCatharanthus species. The principle problem with growing periwinkle isits sensitivity and susceptibility to attack by Phytophthora parasitica.Stem and crown rot caused by Phytophthora parasitica is a common problemon Catharanthus. Symptoms typically are associated with the final stagesof disease development, but infected plants can support a population ofthe pathogen without showing visual symptoms.

Keen and Yoshikawa (in Phytophthora, Erwin et al., eds, 1983) 279-284describe natural mechanisms of resistance to Phytophthora spp. in othercrops. General resistance mechanisms against Phytophthora spp. includestructural features of the host, preformed chemical inhibitors, inducedstructural barriers, hypersensitive reactions and phytoalexins. Keen,Adv. Plant Pathol. 65:35-82 (1982) also suggests that specificresistance to Phytophthora spp. is usually controlled by single hostresistance genes. Monogenetically inherited resistance to differentspecies of Phytophthora has been reported in several crops other thanCatharanthus. Resistance has most often been found to be attributable tosingle, dominant alleles (Umaerus et al., in Phytophtohra, Erwin et al.,eds 1983) 315-326. Colon et al., Euphytica. 66:55-64 (1983) describedresistance to Phytophthora infestans in Solanum spp.

The chemical metalaxyl (which is the active ingredient of theeumycete-specific fungicities Subdue and Ridomil) has been the primaryfungicide used by nursery personnel to control stem and crown rotscaused by Phytophthora spp. as discussed in Ferrin, et al. PlantDisease, Vol. 76, p. 60-63, p. 82-84 (1992). While some control of thediseases is effected by the chemical metalaxyl, Ferrin, supra has foundone isolate of P. parasitica from a southern California nursery whichwas insensitive to metalaxyl. Tolerance to metalaxyl was expressed invivo by the isolate chosen by Ferrin, et al. supra. Furthermore, thismetalaxyl-tolerant isolate appears to be as virulent as sensitivewild-type isolates. For nurseries where plants are grown in containers,widespread failures in disease control would not necessarily be expectedimmediately after the appearance of metalaxyl tolerance because of thelow frequency with which such tolerance appears and the time needed forthe pathogen population to increase and be dispersed. Widespread diseasecontrol failures occur only after a large part of the pathogenpopulation has become tolerant to the pesticide. The rate at which thismetalaxyl-tolerant population becomes established depends largely on thestability of the pesticide tolerance, the selection pressure exerted onthe pathogen population, and the ability of the pathogen to disperse.Because species of Phytophthora and Pythium have been detected inrecycled irrigation water in nurseries in California, the requirementthat commercial nurseries in certain areas of California trap andrecycle all runoff water greatly increases the risk of recyclingfungicide-tolerant populations of these pathogens in those nurseries.Thus, the appearance of metalaxyl tolerance could eventually result incontrol failures if the treatment of recirculated water is notsufficient to eliminate propagules of Phytophthora spp. and theselection pressure due to the continued use of the fungicide metalaxylis maintained.

The continuous use of metalaxyl as the primary means of controllingdiseases caused by Phytophthora increases the likelihood for thedevelopment of insensitivity to metalaxyl (Ferrin et al., supra).History has shown new isolates of P. parasitica insensitive to newfungicides eventually will evolve and since the use of chemicals canhave side effects on people using the chemicals and the environment, itis highly desirable to use a Catharanthus roseus having geneticresistance to Phytophthora spp.

A genetic resistance to Phytophthora in periwinkle, if available, couldbe used to reduce or eliminate the use of the chemical metalaxyl andresult in increased cost efficiencies and environmental safety.

SUMMARY OF THE INVENTION

The present invention relates to a Catharanthus seed, a Catharanthusplant, a Catharanthus variety, a Catharanthus hybrid and a method forproducing a Catharanthus plant.

More specifically, the invention relates to a Catharanthus plant havingresistance to the fungal disease Phytophthora parasitica. The presentinvention also relates to a Catharanthus plant having a moderate to highlevel of resistance to aphids (Myzus persicae) and mites (Tetranychusurticae), and a Catharanthus plant having an increase in total alkaloidcontent.

The present invention further relates to a method of producing thedisclosed Catharanthus plants and seeds by crossing a Phytophthoraresistant plant of the instant invention with another Catharanthusplant. The invention also relates to the transfer of the geneticPhytophthora resistance into genera other than Catharanthus, includingbut not limited to the following genera: Solanum, Capsicum, Eucalyptus,Carica, Ananas, Fragaria, Camellia, Castanea, Persea, and Citrus.

The present invention further relates to a periwinkle plant having alevel of resistance to certain insects, including but not limited toaphids (Myzus persicae) and mites (Tetranychus urticae).

The present invention further relates to a periwinkle plant which has atotal alkaloid content of at least 2.0 percent or greater.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawing,

FIG. 1 is a schematic perspective view of a periwinkle plant alongwherein the numbers 1-10 indicate ratings for various levels ofPhytophthora resistance or sensitivity.

FIGS. 2A, 2B, and 2C show schematically stages of the method ofinoculation of the Catharanthus plant with the Phytophthora inoculum.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide an understanding of several of the terms used in thespecification and claims, the following definitions are provided:

Total alkaloid content--The term `total alkaloid content` is intended torefer to the total alkaloid content expressed as a percent of total dryweight in the plant tissue.

Homogeneous Assemblage--The term `homogeneous assemblage` is intended torefer to a group of seeds or plants which are homogeneous for a givenperiwinkle characteristic. This term is intended to include any seeds orplants which are homogeneous for either of the traits of Phytophthoraresistance or total alkaloid content.

Plant Product--The term `plant product` is intended to refer to plantextracts and plant derived chemical compounds or structures includingalkaloids.

Catharanthus roseus (L) G. Don, also called periwinkle and vinca isprimarily a self-pollinating species. The Catharanthus roseus of thepresent invention reproducibly expresses resistance to Phytophthoraparasitica. We have isolated from our breeding populations atransferrable gene which conveys resistance to Phytophthora. Thisdisease resistance trait has been expressed in many different geneticbackgrounds of periwinkle.

To date, there is no known resistance to Phytophthora in any commercialcultivar of periwinkle except for the present invention. Additionally,there are no known reports of resistance to Phytophthora in anyCatharanthus species, cultivar, in the wild or commercially available.All available cultivars and naturalized collections from the U.S.,Mexico, Central America, Africa and Madagascar, have been tested and noindication of Phytophthora resistance has been found as shown in Tables1 and 2. This previously unknown disease resistance arose from breedingand research efforts which were conducted beginning in 1988.

                  TABLE 1                                                         ______________________________________                                        Phytophthora Resistance in Periwinkle Cultivars                               Cultivar        Resistant:Sensitive Ratio                                     ______________________________________                                        Pretty in Pink  0:1             .sup. (>300).sup.1                            Pretty in White 0:1             (>300)                                        Pretty in Rose  0:1             (>100)                                        Parasol         0:1             (>100)                                        Tropicana Rose  0:1             (>200)                                        Tropicana Bright Eye                                                                          0:1             (>200)                                        Tropicana Pink  0:1             (>200)                                        Tropicana Blush 0:1             (>200)                                        Peppermint Cooler                                                                             0:1             (>100)                                        Grape Cooler    0:1             (>100)                                        Morning mist    0:1             (>100)                                        Pink Panther    0:1               (48)                                        Sahara Madness Bright Eye                                                                     0:1               (48)                                        Polka Dot       0:1               (48)                                        Little Pinkie   0:1             (>300)                                        Little Bright Eye                                                                             0:1             (>300)                                        Little Blanche  0:1             (>300)                                        Little Delicata 0:1               (48)                                        Little Linda    0:1               (48)                                        Inter-variety hybrids.sup.2                                                                   0:1             (>2952)                                       ______________________________________                                         .sup.1 Number in parentheses indicates number of individual plants tested     .sup.2 123 hybrid combinations between commerciallyavailable cultivars an     Goldsmith breeding lines not containing Phytophthora resistance. Includes     line numbers 13043, 13498, 13500, 13509, 13522, 13533, 13534, 13535,          13536, 13639, 13861, 14322, 14590                                        

                  TABLE 2                                                         ______________________________________                                        Phytophthora Resistance in Catharanthus spp.                                  Species   Source         Resistant:Sensitive Ratio                            ______________________________________                                        C. longifolius                                                                          Madagascar (8408).sup.1                                                                      0:1                                                  C. roseus Madagascar (8430)                                                                            0:1                                                            Madagascar (8440)                                                                            0:1                                                            Madagascar (8442)                                                                            0:1                                                            Madagascar (8443)                                                                            0:1                                                            Mexico (13635).sup.2                                                                         0:1                                                  C. trichophyllus                                                                        Madagascar (12914).sup.3                                                                     0:1                                                  ______________________________________                                         .sup.1 Field collection number of R. N. Bowman                                .sup.2 Provided by J. Marsh                                                   .sup.3 Provided by A. Petit                                              

The instant invention is a genetic resistance to the diseasePhytophthora in periwinkle wherein periwinkle plants which are exposedto this disease become infected but there is no spread or movement ofthis disease through the plant. The result is the resistant periwinkleplants stay healthy and do not die after exposure to Phytophthora. Howthis disease resistance gene acts by preventing the spread of thedisease organism within a periwinkle plant is unknown. One possiblemechanism of action is an enhanced alkaloid content within the plant.

The genetic basis for Phytophthora resistance in periwinkle seems toinvolve a single dominant allele. This observation parallelsPhytophthora resistance observed in other crops as described in Umaeruset al. (in Phytophthora, Erwin et al., eds, 1983). Ongoing breedingtrials will help to further characterize the genetic basis forPhytophthora resistance in Catharanthus. When the disease resistancegene is incorporated into different genetic backgrounds of periwinkle,the disease resistance characteristic is transferred into these geneticbackgrounds.

Once true-breeding resistant lines are developed that also possesshorticulturally valuable traits (ex. compact habit, desirable colors),then open pollinated resistant seed from these lines can be marketed.Also possible is the use of a true-breeding resistant line as one of theparents in F₁ hybrid seed production resulting in an F₁ hybrid whichexpresses Phytophthora resistance. Additionally, transferring thePhytophthora resistance gene of the present invention into crops otherthan Catharanthus may reduce the damage caused by Phytophthora in thosecrops.

As used herein, the term "plant" includes plant cells, plantprotoplasts, plant cells of tissue culture from which periwinkle plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

Tissue culture of periwinkle is described in van der Heijden et at."Cell and Tissue Cultures of Catharanthus roseus (L.) G. Don: ALiterature Survey", Plant Cell, Tissue and Organ Culture 18:231-280(1989), incorporated herein by reference. As described in this article,the presence of the therapeutically valuable cytotoxic alkaloidsresulted in Catharanthus becoming one of the major fields of interest inmodern plant cell biotechnology. The low yields of these dimeric indolealkaloids (approximately 0.0005%) and the subsequent high price of themwere major motives to study the possibilities for the production ofthese alkaloids by cell and tissue cultures. The first efforts in thistissue culture with periwinkle date from approximately 25-30 years ago.Thus, another aspect of this invention is to provide for cells whichupon growth and differentiation produce the disease resistance toPhytophthora in periwinkle.

In addition to providing resistance to Phytophthora, the transfer of thedominant resistance gene to different genetic backgrounds has producedwhat we believe are the associated characteristics of increased totalalkaloid content expressed as a percent of total dry weight in the plantand increased tolerance or resistance to certain insects, includingaphids and mites. It is believed that the elevated level of totalalkaloid content may be directly associated with the increased plantresistance to aphids and mites and may also be somewhat associated withthe level of Phytophthora resistance. The important traits of increasedtotal alkaloid content and insect resistance are additional objects ofthe present invention.

Eilert et al., as discussed in Plant Physiol., 126:11 (1986); Arch.Biochem., Biophys., 254:491 (1987) were able to induce alkaloidaccumulation in a cell suspension culture of C. roseus upon treatment ofthe cultures with homogenates of various fungi, thus further suggestinga relationship between alkaloid production and potential fungatresistance. Nef et al., Plant Cell Reports 10:26-29 (1991), were alsoable to stimulate major alkaloid production in Catharanthus roseus cellswith an extract of the fungus Pythium vexans. Finally, vanDam, et al.,Oecologia 95:425-430 (1993) found that indole alkaloid production inCatharanthus roseus was not inducible by mechanical leaf damage alone.The Phytophthora resistance gene of the instant invention is stronglyimplicated in the modulation of alkaloid synthesis and activity inCatharanthus. This activity, however, does not preclude other possibleaspects of the resistance phenomenon.

Development of the Phytophthora resistance gene in Catharanthus hasprovided a straightforward means of providing non-pesticide protectionto an important horticultural crop. Use of the gene imparts protectionfrom ubiquitous Phytophthora infection and may aid in protection fromother fungal and insect pathogens. Additionally, the gene is both userand environment friendly by reducing or eliminating the amounts ofpesticides necessary to maintain the crop. Protection from Phytophthoraas well as other potential broad spectrum protection makes theresistance gene potentially valuable in other crops. The annual dollarloss from Phytophthora infections is enormous. Phytophthora cinnarnomi,alone, has nearly 1,000 hosts as discussed in Zentmeyer, Monogr. 10, Am.Phytopathol. Soc. 96 pp. (1980), many of which are important cropplants. Phytophthora infestans, the cause of the late blight ofpotatoes, which produced the great potato famine of 1846-47, resulted inthe starvation of about 800,000 people. The disease is still a seriousproblem today. Non-pesticide reduction or elimination of Phytophthoralosses would be of much significance to both food and ornamental crops.Any new source of resistance to either aerial or root Phytophthoradiseases is potentially very valuable.

EXAMPLES

The following examples are provided to further illustrate the presentinvention and are not intended to limit the invention beyond thelimitations set forth in the appended claims.

EXAMPLE 1 In Vitro Growth and Inoculation with the Phytophthora Pathogen

In vitro culture of Phytophthora parasitica, the causative agent of wiltin Catharanthus, is described in Gill, et at., (1977). The fungusreadily grows on V-8 juice agar. Isolation and identification of strainsof the fungus were accomplished by methods known to those skilled in theart. In testing for resistance to the fungus, we used the 12 differentfungal isolates shown in Table 3 including pathogens obtained fromcommercial nurseries and plantings experiencing severe fungal attack.Isolates were obtained from Ferrin et al., as discussed in Ferrin etal., supra. Periwinkle plants were grown from seed using standardmethods commonly known to those in the nursery trade. Plants were grownto the five-plus node stage (as shown in FIG. 1) before inoculation. Forcomparisons of pathogenicity between the Phytophthora isolates,individual periwinkle plants were cloned via stem cuttings prior toinoculations.

Axenic Phytophthora cultures were used to prepare inoculation capsulesas shown in FIGS. 2A, 2B, and 2C. A clear plastic drinking straw (13)was used to punch agar explants (12) of the fungus from the culture dish(11). The agar explant (12) was then positioned into a short inoculationcapsule (15) where a 15 mm segment of the plastic drinking straw (13)was sealed at one end with a 6 mm. diameter spherical glass bead (14).The surface of the fungal mycelium was placed away from the bead.

Periwinkle seedlings were prepared by cutting the upright stem betweenthe fourth and fifth nodes with a clean razor blade. As shown in FIG. 1,an inoculation capsule (15) was then placed on the stem stump and gentlypressed downward, thus forcing the fungal mycelium against the exposedstem surface. Controls were prepared in a similar manner, but withoutthe fungal isolate.

The inoculated plants were then grown in a greenhouse using normalmethods known to those skilled in the art. After approximately threeweeks, the plants were then scored for the degree of movement of thefungus in the plant tissues,

                  TABLE 3                                                         ______________________________________                                        Sources of Phytophthora parasitica cultures.sup.1                             Isolate                                                                             Source                                                                  ______________________________________                                        A     Premier Color Nursery, Fallbrook, CA                                    B     Premier Color Nursery, Fallbrook, CA                                    C     Premier Color Nursery, Fallbrook, CA                                    D     Premier Color Nursery, Fallbrook, CA                                    E     Premier Color Nursery, Fallbrook, CA                                    F     Premier Color Nursery, Fallbrook, CA                                    G     Premier Color Nursery, Fallbrook, CA                                    H     Summerlin Project, Summerlin Parkway, Las Vegas, NV                     I     Summerlin Project, Summerlin Parkway, Las Vegas, NV                     J     The Lakes Country Club, home garden,                                          Palm Desert, CA                                                         K     Color Spot Nursery, Fallbrook, CA                                       L     D. Ferrin, see Ferrin et al., 1992                                      ______________________________________                                         .sup.1 Identity of all 12 isolates as P. parasitica was confirmed by Dr.      J. Mitchell, Department Plant Pathology, University of Florida,               Gainsville.                                                              

Example 2 Assessment of Infection--Definition of Resistance

The rating scale 1-10 as described in Table 4 and schematically shown inFIG. 1 was used to measure the degree of infection and extent ofmovement of the pathogen through intact tissues of the inoculated plant.The degree of infection for each seedling in a family was determined aswere the ratios of resistant to sensitive progeny as identified inTables 1, 2, 5, 6 and 7. Three weeks after inoculation, the extent oftissue necrosis was assessed using the rating scale of Table 4 andFIG. 1. Plants that were able to arrest the movement of the funguswithin the uppermost portion of the top internode were consideredresistant; additionally, plants showing partial necrosis at theuppermost node but without continued movement below the top-most nodewere considered resistant if the fungus did not continue downwardnearing the second node. Resistance in Catharanthus to Phytophthoraparasitica was defined as the ability to arrest the movement of thepathogen at or near the first node below the infection site. Incontrast, sensitivity was defined as the inability to arrest themovement of the pathogen ultimately resulting in death of the plant.

                  TABLE 4                                                         ______________________________________                                        Rating Scale for Resistance Testing                                           Ratings of 5-10 are considered resistant.                                     Ratings of 1-4 are considered sensitive or susceptible.                       Rating                                                                        Scale Description of Category                                                 ______________________________________                                              Also: See FIG. 1 for Schematic Representation of Rating                       Scale                                                                   10    Clear Abscission zone formed well above uppermost                             node; no sign of darkening below abscission zone;                             abscission zone forming quickly.                                        9     Necrosis clearly stopped well above uppermost node; no                        abscission zone but no darkening (or continuation of                          darkening) well above uppermost node.                                   8     Necrosis stopped in crown at or just above uppermost                          node; no sign of infection in uppermost leaves.                         7     One or both uppermost leaves chlorotic or clearly infect-                     ed but not rapidly dying. The infecfion has been stopped                      at the node.                                                            6     Infection present in uppermost portion of second inter-                       node, appearing arrested just below the top node.                       5     Compared to the sensitive control plants progression of                       necrosis is occurring much more slowly. Necrosis                              arrested within the second internode, with no further                         movement, even after prolonged time.                                    4     Upper node dead, darkening of lower axils; several nodes                      clearly affected. No apparent arrest of the disease.                    3     Two or more nodes dead; other nodes showing infection.                  2     Some green in lower stem but death will occur in a few                        days.                                                                   1     Rapid and complete death, as in non-resistant controls.                 ______________________________________                                    

Example 3 Lack of Phytophthora Resistance in Existing CatharanthusCultivars

All commercially available cultivars developed by public institutionsand/or companies other than Goldsmith Seeds, Inc. were screened forPhytophthora resistance using the aforementioned methods. Results of thetests are shown in Table 1 and indicate that no commercial cultivar isresistant to Phytophthora. No commercially available cultivar iscurrently claimed by its manufacturer or distributor to be Phytophthoraresistant. Chase and King, Grower Talks 57:63-69 (1993) have suggestedthat some cultivars are more susceptible to Phytophthora than others;they, however, found that all cultivars they tested were susceptible toP. parasitica.

Example 4 Lack of Phytophthora Resistance in Wild Catharanthus spp.

Assessions of Catharanthus spp. germplasm were also tested forPhytophthora resistance using the aforementioned methods of Examples 1and 2. Wild germplasm of Catharanthus is difficult to obtain due tolimited native distributions (Madagascar) and widespread destruction ofits native habitat. Germplasm was collected in Madagascar ofC.longifolius and C. roseus. Also representative germplasm ofC.trichophyllus from Annik Petit (CNRS, Institut des Sciences Vegetales,France) was obtained. Non-resistance in these assessions is summarizedin Table 2. Clearly, wild species of Catharanthus are not Phytophthoraresistant.

Example 5 New Resistant Lines

As shown in Table 5, in the process of screening several hundred speciesx species, cultivar x species, and cultivar x cultivar Catharanthushybrids for Phytophthora resistance in June, 1992, three experimentalhybrid lines, 13516, 13517 and 13518 were identified that expressed goodresistance. At that time, this resistance was the only resistance whichwe had ever found, despite concurrent extensive testing of all availablenative and naturalized collections and all existing cultivar germplasm.Further, the lines were not true breeding for resistance since theysegregated both resistant and sensitive individuals as shown in Table 5.Subsequent analysis of pedigrees also shown in Table 5 suggested thatthe resistance may be the consequence of a single dominant (R) allele.Genetic analysis was complicated by the fact that parents in precedinggenerations had not been previously tested for resistance but had beendiscarded after the crosses were completed.

                                      TABLE 5                                     __________________________________________________________________________    Pedigree of lines 13516, 13517, and 13518,                                    in which Phytophthora resistance was initially identified                     8424-3.sup.a × 10132-1.sup.b =                                                      11461                                                             11461-1.sup.c × SELF =                                                              12669                                                                         12669-1.sup.c × SELF =                                                              13516 (segregating for resistance.sup.d)                          12669-3.sup.c × SELF =                                                              13517 (segregating for resistance.sup.d)                          12669-4.sup.c × SELF =                                                              13518 (segregating for resistance.sup.d)              11461-1.sup.c × 11969-12.sup.b =                                                    12825                                                                         12825-1.sup.c × SELF =                                                              14681 (all sensitive)                                             12825-2.sup.c × SELF =                                                              14680 (all sensitive)                                 11892-5.sup.b × 11461-1.sup.c =                                                     12819                                                                         12819-1.sup.c × 13536-51.sup.c =                                                    14282 (all sensitive)                                 __________________________________________________________________________     .sup.a Field collection of R. N. Bowman, actual resistance unknown; plant     was discarded after breeding crosses were made.                               .sup.b Cultivar PrettyIn-White, not known to contain any resistance; see      Table 1.                                                                      .sup.c Actual resistance unknown; plant was discarded after breeding          crosses were made.                                                            .sup.d See Tables 67 for continuation of these lines.                    

Pedigree analysis of the three lines carrying resistance (13516, 13517,13518) indicated they shared in common a single plant (8424-3). Thissingle plant had been considered a mutant form because of its light bluecolor and extreme infertility. We had never before seen the flower colorin any wild species, cultivar or hybrid; further, the plant did not fitthe key of any wild species as discussed in Veyret, CatharanthusAlkaloids, (1974). The initial motivation for using the plant in abreeding program was to capture the mutant color, remove it from itsinfertile background and use it for development of a new cultivar color.

Additional crossing generations were undertaken to better understand thegenetic basis of the resistance geneo

Example 6 Genetic Segregation of Resistance

Individual plants from line numbers 13516, 13517, and 13518 were selfed;progenies in the next generation were checked for segregation ofresistance (see Table 5). Additionally, as shown in Table 6, individualsfrom 13516, 13517, 13518 were also outcrossed to known sensitive lines.The F₁ progenies from these out crosses were also assessed forresistant: sensitive ratios.

The presence of sensitive progeny in selfs of phenotypically resistantindividuals confirms that resistance is controlled by a dominant allele.Segregating ratios in selfs and F₁ generations confirm that resistanceis monogenically controlled by two alleles, (R) for Phytophthoraresistant, and (r) for Phytophthora sensitive. Table 7 indicates that F₂segregations further substantiate the genetic basis for Phytophthoraresistance in Catharanthus. In combination, Tables 5-7 furtherdemonstrate the genetic basis for the resistance gene as a singledominant allele.

                                      TABLE 6                                     __________________________________________________________________________    F.sub.1 and Self Crosses Involving Resistant Phenotype Individuals                                     Segregating                                          Resistant                Progeny Ratio                                        Phenotype                                                                           Female Male   Cross                                                                              (Resistant:                                          Individual                                                                          Parent Parent Number                                                                             Sensitive)                                                                            Conclusion                                   __________________________________________________________________________    13516-2                                                                             13516-2 ×                                                                      SELF = 14602                                                                              (3:1.sup.a)                                                                           13516-2 is heterozygous, Rr                        14590-9.sup.b ×                                                                13516-2 =                                                                            14800                                                                              (1:1.sup.a)                                                                           "                                                  13534-10.sup.b ×                                                               13516-2 =                                                                            14705                                                                              (1:1.sup.a)                                                                           "                                                  13522-9.sup.b ×                                                                13516-2 =                                                                            14712                                                                              (1:1.sup.a)                                                                           "                                            13516-8                                                                             13516-8 ×                                                                      SELF = 14606                                                                              (1:0.sup.a)                                                                           13516-8 is homozygous, RR                          12974-5.sup.b ×                                                                13516-8 =                                                                            14662                                                                              (1:0.sup.a)                                                                           "                                                  13522-6.sup.b ×                                                                13516-8 =                                                                            14663                                                                              (1:0.sup.a)                                                                           "                                                  13533-2.sup.b ×                                                                13516-8 =                                                                            14664                                                                              (1:0.sup.a)                                                                           "                                                  13536-41.sup.b ×                                                               13516-8 =                                                                            14667                                                                              (1:0.sup.a)                                                                           "                                            13516-17                                                                            13516-17 ×                                                                     SELF = 14605                                                                              (3:1.sup.c)                                                                           13516-17 is heterozygous, Rr                       13522-6.sup.b ×                                                                13516-17 =                                                                           14669                                                                              (1:1.sup.a)                                                                           "                                            13516-17                                                                            13534-26.sup.b ×                                                               13516-17 =                                                                           14671                                                                              (1:1.sup.a)                                                                           "                                            (cont'd)                                                                            13535-50.sup.b ×                                                               13516-17 =                                                                           14672                                                                              (1:1.sup.a)                                                                           "                                                  13536-41.sup.b ×                                                               13516-17 =                                                                           14673                                                                              (1:1.sup.a)                                                                           "                                                  13534-6.sup.b ×                                                                13516-17 =                                                                           14703                                                                              (1:1.sup.a)                                                                           "                                                  13516-17 ×                                                                     13500-18.sup.b =                                                                     14711                                                                              (1:1.sup.a)                                                                           "                                                  13861-1.sup.b ×                                                                13516-17 =                                                                           14790                                                                              (1:1.sup.a)                                                                           13516-17 is heterozygous Rr                        13043-3.sup.b ×                                                                13516-17 =                                                                           14844                                                                              (1:1.sup.a)                                                                           "                                            13518-43                                                                            13518-43 ×                                                                     SELF = 14912                                                                              (3:1.sup.a)                                                                           13518-43 is heterozygous, Rr                       14322-39.sup.b ×                                                               13518-43 =                                                                           15060                                                                              (1:1.sup.a)                                                                           "                                            13518-49                                                                            13518-49 ×                                                                     SELF = 14900                                                                              (3:1.sup.a)                                                                           13518-49 is heterozygous, Rr                       14322-18.sup.b ×                                                               13518-49 =                                                                           15038                                                                              (1:1.sup.a)                                                                           "                                                  13518-49 ×                                                                     13500-11.sup.b =                                                                     15224                                                                              (1:1.sup.a)                                                                           "                                                  13518-49 ×                                                                     13509-1.sup.b =                                                                      15226                                                                              (1:1.sup.a)                                                                           "                                            13518-59                                                                            13518-59 ×                                                                     SELF = 14919                                                                              (1:0.sup.a)                                                                           13518-59 is homozygous, RR                         14322-46.sup.b ×                                                               13518-59 =                                                                           15064                                                                              (1:0.sup.a)                                                                           "                                                  14322-1.sup.b ×                                                                13518-59 =                                                                           15065                                                                              (1:0.sup.a)                                                                           "                                                  14322-36.sup.b ×                                                               13518-59 =                                                                           15385                                                                              (1:0.sup.a)                                                                           "                                            13518-63                                                                            13518-63 ×                                                                     SELF = 14919                                                                              (1:0.sup.a)                                                                           13518-63 is homozygous, RR                         14322-23.sup.b ×                                                               13518-63 =                                                                           15039                                                                              (1:0.sup.a)                                                                           "                                                  13639-3.sup.b ×                                                                13518-63 =                                                                           15040                                                                              (1:0.sup.c)                                                                           "                                            13518-69                                                                            13518-69 ×                                                                     SELF = 14902                                                                              (3:1.sup.a)                                                                           13518-69 is heterozygous, Rr                       14322-12.sup.b ×                                                               13518-69 =                                                                           15042                                                                              (1:1.sup.c)                                                                           "                                                  13518-69 ×                                                                     13498-3.sup.b =                                                                      15227                                                                              (1:1.sup.a)                                                                           "                                                  13518-69 ×                                                                     13500-11.sup.b =                                                                     15228                                                                              (1:1.sup.a)                                                                           "                                                  13518-69 ×                                                                     13509-1.sup.b =                                                                      15229                                                                              (1:1.sup.a)                                                                           "                                            __________________________________________________________________________     .sup.a Significant ratio based on Chi Square, P < .05.                        .sup.b Known to be sensitive, from a background that has never segregated     any resistance, see Table 1.                                                  .sup.c Sample size too small for significance test.                      

                                      TABLE 7                                     __________________________________________________________________________    F.sub.2 Phenotypic Segregation for Resistance in                              Lines Derived from Populations 13516, 13517, and 13518.                       __________________________________________________________________________    14590-9.sup.b × 13516-2.sup.a =                                                     14800        F.sub.2 = 15808                                                  progeny segregated 1:1                                                                     expected 3:1 (RES:SEN)                                           (RES:SEN), SEN progeny                                                                     observed 3:1 (72:21), significant at p <<.05                     discarded; RES progeny                                                        selfed for F.sub.2                                                13522-6.sup.b × 13516-17.sup.a =                                                    14669        F.sub.2 = 15770                                                  progeny segregated 1:1                                                                     expected 3:1 (RES:SEN)                                           (RES:SEN), SEN progeny                                                                     observed 3:1 (126:32), significant at p <<.05                    discarded; RES progeny                                                        selfed for F.sub.2                                                13533-2.sup.b × 13516-8.sup.a =                                                     14664        F.sub.2 = 15795                                                  progeny segregated 1:0                                                                     expected 3:1 (RES:SEN)                                           (RES:SEN), RES progeny                                                                     observed 3:1 (216:70), significant at p <<.05                    selfed for F.sub.2                                                __________________________________________________________________________     .sup.a See Table 6 for determination of genotype.                             .sup.b Known to be sensitive, from a background that never segregates         resistance, see Table 1.                                                 

Example 7 Phytophthora Resistant Open--Pollinated Cultivars

As previously discussed, all existing commercial periwinkle cultivarsare produced as open-pollinated crops. Flowers are primarily selfpollinated in production fields or greenhouses and the resulting seed isharvested and sold. Open pollinated crops, including periwinkle, must betrue breeding (homozygous) for desirable traits in order for expressionof uniform continuity. Homozygous resistant (RR) lines have beendeveloped which segregate only the resistant phenotype in all succeedingopen-pollinated generations. The homozygous resistant lines nowincorporate other homozygous horticulturally desirable traits including,but not limited to, plant habit, flower colors and flower size. Usingconventional cross breeding methods, we have now crossed thePhytophthora resistance gene into all of the commercially significantperiwinkle cultivars listed in Table 1 including the "Little", "Cooler","Pretty In", and "Tropicana" series. Both the expression and segregationof the resistance gene in these diverse backgrounds were transferred aswe had expected. This broad genetic base was used in the development ofnew Phytophthora resistant cultivars.

Example 8 Phytophthora Resistant F₁ Hybrid Cultivars

Even though all currently available commercial periwinkle cultivars areproduced as open-pollinated crops, Phytophthora resistant F₁ hybridsalso can be produced. In F₁ hybrids, a female line, incapable ofpollinating itself either because it lacks pollen or is manuallyemasculated, is pollinated with pollen from another pollen-producing("male") line. The resulting F₁ hybrid benefits from hybrid vigor andexpresses aspects of both the pollen parent and female genomes. Otherthan periwinkle, a very large proportion of both the flower andvegetable seed, now sold is F₁ hybrid seed.

Periwinkle lines have been produced which are suitable for use asfemales in F₁ hybrid seed production. Because Phytophthora resistance ismonogenically controlled by a dominant allele, either the male, femaleor both can provide Phytophthora resistance which will be expressed inthe F₁ hybrid generations. Such a production scheme has resulted in newF₁ hybrid cultivars that are uniformly Phytophthora resistant.

Example 9 Additional Resistance to Other Pests including Aphids andMites

Long term breeding programs for development of new periwinkle cultivarshave provided ample opportunity to observe performance of existingcultivars and breeding lines with respect to sensitivity to commongreenhouse pests including aphids (Myzus persicae) and mites(Tetranychus urticae). Table 8 indicates qualitative differences insusceptibility of selected lines and cultivars to these commongreenhouse pests. Interestingly, lines selected for resistance toPhytophthora are less prone to attack by greenhouse pests such as aphidsand inites. Conversely, lines known to be Phytophthora sensitive, aremore prone to attack by aphids and mites. As shown in Table 8, line13812 which was selected for extreme Phytophthora sensitivity is alsoextremely prone to aphid and mite infestations. Expression of thePhytophthora resistance gene of the instant invention appears to bestrongly correlated with enhanced protection from other pests including,but not limited to, aphids and mites.

                  TABLE 8                                                         ______________________________________                                        Comparative.sup.a Mite and Aphid Resistance in                                Selected Catharanthus Cultivars and Breeding Lines.sup.b                      Cultivar or                                                                            Phytophthora                                                                             Aphid Resistance                                                                           Mite Resistance                              Line Number                                                                            Resistant  Level.sup.c  Level.sup.c                                  ______________________________________                                        13500    No         Average      -                                            14322    No         Average      Average                                      13812    No         --           --                                           15620    Yes        Average      +                                             8941    Yes        ++           ++                                           15667    Yes        ++           ++                                           ______________________________________                                         .sup.a Based on visual observations of plants grown in controlled             greenhouse environment.                                                       .sup.b See Table 9 for description of line numbers.                           .sup.c -- is very sensitive, - is sensitive, + is somewhat resistant, ++      is very resistant.                                                       

Example 10 Evaluation of Resistant Phenotypes--Possible Mechanism(s) ofAction

As discussed prevously, Catharanthus species are well known for theirproduction of alkaloids. Table 9 indicates the relationship betweentotal alkaloid content and expression of Phytophthora resistance inselected Catharanthus lines. Line 15667, selected for Phytophthoraresistance, contains more than 3.6 times the consensus level of totalalkaloids reported for any other Catharanthus species or cultivar. Line15667 is also highly resistant to aphids and mites as shown in Table 8.Indirect evidence thus indicates that the mode of action of thePhytophthora resistance gene involves enhanced alkaloid productionand/or activity. Other lines such as 15614 and 15620 in Table 9, do nothave elevated total alkaloid levels, yet still express Phytophthoraresistance, thus suggesting that qualitative changes in alkaloidcomposition, rather than total alkaloid content, may have effected theresistance phenotype, The Phytophthora resistance gene of the instantinvention is strongly implicated in the modulation of alkaloid synthesisand activity in Catharanthus. This activity, however, does not precludeother possible aspects of the resistance phenomenon.

Example 11 Transformation of Other Crop Species with the Resistance Geneto Provide Pest Resistance

The direct applicability of using the Phytophthora resistance gene ofthe present invention in transformation of other crops species sensitiveto Phytophthora diseases is significant. Transformation methods nowapplied to many plant species (cf. Robinson and Riroozabady, ScientiaHorticulturae 55:83-99, 1993) enable the movement of desirable genesfrom a source (in this case, Phytophthora resistant Catharanthus) to atarget species. Using currently available molecular methods known tothose in the art, the gene is sequenced, cloned and inserted in otherspecies where its expression is valuable. Expression of the resistancegene in crops now sensitive to aerial Phytophthora diseases includingpalms, Bougainvillea and other ornamentals provide a new source ofnon-pesticide protection. At first glance, one might suspect thatheightened alkaloid levels (and thus Phytophthora resistance) mightinterfere with food crops. While this situation is possible, potatoesprovide a good example of how food crops will benefit fromtransformational insertion of our resistance gene; potato stems andleaves are already toxic due to high levels of alkaloids they contain,yet tubers produced by the plants are edible. Thus, altered alkaloidexpression, accomplished through transformation with the resistance geneof the present invention may impart additional non-pesticide cropprotection without affecting the food quality of the crop.

Selected genes have now been isolated and cloned from Catharanthusroseus. Meijer, et al., Plant Mol. Biol., 22:379-383 (1993),incorporated herein by reference, isolated cytochrome P-450 cDNA clonesfrom C. roseus using standard methods known to those skilled in the art.Goddijin, et al., Plant Mol. Biol. 22:907-912 (1993) incorporated hereinby reference, isolated a tryptophan decarboxylase gene from C. roseus,and used this same gene as a selectable marker in Agrobacterium-mediatedtobacco leaf disc transformation experiments. They were able to recovermature transformed tobacco plants expressing the Catharanthus gene.Methods are, thus, currently available to those skilled in the art, fortransformations using our Phytophthora resistance gene in other cropspecies.

Example 12 Commercial Alkaloid Production Use of the Resistance Gene toModulate Alkaloid Quantity and Quality

Selection for resistance to Phytophthora has resulted in an increasedlevel of alkaloids in selected lines. Importantly, however, it should benoted that these lines were not specifically selected for alkaloidcontent or quality. Since it is now apparent that the resistance genedirectly influences the total amount of alkaloids produced, as shown inTable 9, one can intentionally select for increased alkaloid content byscreening lines that contain the resistance gene. Yoder and Mahlberg Am.J. Bot;., 63:1167-1173 (1976) suggested that the high concentration("1.5% ") of indole alkaloids in leaves of Catharanthus roseus and thefact that they are mainly found in specialized cells indicate that theplant stores indole alkaloids constitutively and probably has alreadyevolved mechanisms to overcome auto-intoxication. It is expected thatelevated alkaloid contents can be attained which exceed those reportedin Table 9; further, selection for specific alkaloids rather than totalalkaloid content results in an effective means of improving alkaloidproduction in Catharanthus. Currently, we are not aware of any U.S.patents covering alkaloid synthesis genes in Catharanthus; additionally,the synthetic pathways leading to the significant alkaloids includingcatharanthine, vindoline, vinblastine and vincristine are only reportedfrom Catharanthus. Quantitatively, Cathanthine, vindoline andanhydrovinblastine are the alkaloids in greatest abundance inCatharanthus as discussed in Balsevich and Hogge, J. Nat. Prod. 51:1173-1177. Field grown plants of Catharanthus roseus are the onlycommercial soume (at present) for the production of anti-cancertherapeutic alkaloids vinblastine and vincristine. Given the dollarvalue of periwinkle alkaloids, even small improvements in alkaloidquantity or quality can translate to enormous economic potential. Over along period of time, numerous attempts have been made to use tissueculture methods for in vitro production of alkaloids. Economicefficiency of these in vitro methods has not been achieved, in part dueto the low amounts of alkaloids produced. Use of the Phytophthoraresistance gene to increase levels of alkaloid production and/or qualitycan make in vitro alkaloid production feasible. For this reason, theresistance gene can be used in whole plants, plant organs, tissues,cells, protoplasts or cell extracts for any aspect of Catharanthusalkaloid production. Plants containing the genetic resistance allele ofthe present invention are grown under field production conditions toobtain the desired alkaloid production.

                  TABLE 9                                                         ______________________________________                                        Total Alkaloid Content.sup.a (as percent of total                             dry weight) of Selected Catharanthus Lines and Cultivars                             Alkaloid                                                               Line   Content                                                                Number (%)      Line Description                                              ______________________________________                                        --     0.37.sup.b                                                                              Catharanthus roseus, average of five leaf                                    samples as reported in Farnsworth, Lloydia                                    24:105-139 (1961)                                             --     1.5.sup.b                                                                               Catharanthus roseus, as reported in Yoder                                    and Mahlberg, Am. J. Bot., 63:1167-1173                                       (1976)                                                        --     0.57.sup.b                                                                              Catharanthus roseus, as reported in Jones                                    (1976), U.S. Pat. No. 3,932,417.                              --     1.98.sup.b                                                                              Catharanthus roseus, as reported in vanDam,                                  Oecologia 95:425-430 (1993)                                   13500  1.48     Commercial Cultivar "Morning Mist"                            14322  1.70     Goldsmith line, sensitive to Phytophthora                     13812  0.69     Goldsmith line, selected for extreme                                          sensitivity to Phytophthora                                   15614.sup.c                                                                          1.72     Goldsmith line, selected for resistance                                       to Phytophthora                                               15620.sup.c                                                                          1.71     Goldsmith line, selected for resistance                                       to Phytophthora                                               15655.sup.c                                                                          2.70     Goldsmith line, selected for resistance                                       to Phytophthora                                               15667.sup.c                                                                          6.26     Goldsmith line, selected for resistance                                       to Phytophthora                                               ______________________________________                                         .sup.a See footnote on page w.                                                .sup.b See footnote on page w.                                                .sup.c See footnote on page w.                                                .sup.a Leaf samples were taken from eight fieldgrown plants; extractions      and analyses performed by Centre Analytical Labs, Inc., State College, PA     Extractions performed using a method based on Endo et al., Planta Medica,     53:479-482 (1987) incorporated herein by reference. Dried leaf material       was ground and extracted with a suitable solvent (such as methanol), the      solvent was taken to dryness, the residue reextracted with dilute acid,       the solution adjusted to pH 10, then phase partitioned with a siutable        solvent (such as ethyl acetate). The solvent fraction was then evaporated     yielding to total alkaloid sample.                                            .sup.b As noted by Fransworth, Lloydia, 24:105-139 (1961), total alkaloid     amounts reported in the literature can be very ambiguous, due to              differences in growth conditions and extraction methodologies. Jones          (1976) U.S. Pat. No. 3,932,417 incorporated herein by reference,              discussed, in detail, extraction methods for Catharanthus roseus              alkaloids. The highest reported level in leaves (1.98%) is the citation       indicated above. For comparative purposes, I used a consensus value of        1.7% for the total content of leaf alkaloids according to the literature      for Catharanthus roseus.                                                      .sup.c Line segregating for Phytophthora resistance; it is unknown if the     plants sacrificed for sampling were phenotypically resistant or sensitive                                                                              

DEPOSIT INFORMATION

Periwinkle seeds have been placed on deposit with the American TypeCulture Collection (ATCC), Rockville, Md. 20852, under Deposit AccessionNumber 75636 on Jan. 14, 1994.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

What is claimed is:
 1. A Catharanthus seed containing a geneticresistance to Phytophthora, wherein said seed has a pedigree whichincludes the plant 8424-3.
 2. The Catharanthus seed of claim 1, whereinsaid seed contains a dominant allele for resistance to Phytophthoraparasitica.
 3. A Catharanthus plant produced by growing the seed ofclaim
 1. 4. Pollen of the plant of claim
 3. 5. An ovule of the plant ofclaim
 3. 6. A tissue culture comprising regenerable cells of the plantof claim 3, wherein said plant contains a genetic resistance toPhytophthora.
 7. A method for producing F₁ hybrid Catharanthus seedcomprising crossing a first parent Catharanthus plant with a secondparent Catharanthus plant and harvesting the resultant F₁ hybridCatharanthus seed, wherein said first or second parent Catharanthusplant is the Catharanthus plant of claim
 3. 8. The method of claim 7,wherein said Catharanthus plant is the female plant.
 9. The method ofclaim 7, wherein said Catharanthus plant is the male plant.
 10. A firstgeneration (F₁) hybrid Catharanthus plant produced by growing saidhybrid Catharanthus seed of claim
 7. 11. A Catharanthus plant accordingto claim 3, having a total alkaloid content of the leaf tissue of about2.0% or greater.
 12. A Catharanthus plant according to claim 3, whereinsaid total alkaloid content of the leaf tissue is between about 2.0% andabout 3.0%.
 13. A Catharanthus plant according to claim 3, wherein saidtotal alkaloid content of the leaf tissue is between about 3.0% andabout 4.0%.
 14. A Catharanthus plant according to claim 3, wherein saidtotal alkaloid content of the leaf tissue is between about 4.0% andabout 5.0%.
 15. A Catharanthus plant according to claim 3, wherein saidtotal alkaloid content of the leaf tissue is between about 5.0% andabout 6.0%.
 16. A Catharanthus plant according to claim 3, wherein saidtotal alkaloid content of the leaf tissue is between about 6.0% andabout 6.5%.
 17. A Catharanthus plant product derived from asubstantially homogeneous assemblage of Catharanthus plants having atotal alkaloid content of the leaf tissue of about 2.0% to about 6.5%,said plants having a pedigree which includes the plant 8424-3.
 18. ACatharanthus plant product derived from a substantially homogeneousassemblage of Catharanthus plants according to claim 17, wherein saidtotal alkaloid content is between about 2.0% and about 3.0%.
 19. ACatharanthus plant product derived from a substantially homogeneousassemblage of Catharanthus plants according to claim 17, wherein saidtotal alkaloid content is between about 3.0% and about 4.0%.
 20. ACatharanthus plant product derived from a substantially homogeneousassemblage of Catharanthus plants according to claim 17, wherein saidtotal alkaloid content is between about 4.0% and about 5.0%.
 21. ACatharanthus plant product derived from a substantially homogeneousassemblage of Catharanthus plants according to claim 17, wherein saidtotal alkaloid content is between about 5.0% and about 6.0%.
 22. ACatharanthus plant product derived from a substantially homogeneousassemblage of Catharanthus plants according to claim 17, wherein saidtotal alkaloid content of about 6.0% and about 6.5%.
 23. A Catharanthusvariety consisting of a substantially homogeneous assemblage ofCatharanthus plants having a total alkaloid content of about 2.0% toabout 6.5%, said plants having a pedigree which includes the plant8424-23.
 24. A Catharanthus variety consisting of a substantiallyhomogeneous assemblage of Catharanthus plants according to claim 23,wherein said total alkaloid content is between about 2.0% and 3.0%. 25.A Catharanthus variety consisting of a substantially homogeneousassemblage of Catharanthus plants according to claim 23, wherein saidtotal alkaloid content is between about 3.0% and 4.0%.
 26. ACatharanthus variety consisting of a substantially homogeneousassemblage of Catharanthus plants according to claim 23, wherein saidtotal alkaloid content is between about 4.0% and 5.0%.
 27. ACatharanthus variety consisting of a substantially homogeneousassemblage of Catharanthus plants according to claim 23, wherein saidtotal alkaloid content is between about 5.0% and 6.0%.
 28. ACatharanthus variety consisting of a substantially homogeneousassemblage of Catharanthus plants according to claim 23, wherein saidtotal alkaloid content is between about 6.0% and 6.5%.
 29. A method forproducing a periwinkle plant having a total alkaloid content of the leaftissue of 2.0% to about 6.5% comprising the steps of:a) planting inpollinating proximity seeds of a periwinkle genotype containing agenetic resistance to Phytophthora, wherein said seeds have a pedigreewhich includes the plant 8424-3, and another periwinkle genotype; b)cultivating periwinkle plants resulting from said seeds until saidplants bear flowers; c) emasculating the male flowers of the plants ofeither periwinkle genotype; d) allowing cross pollination to occurbetween said periwinkle genotypes; e) harvesting seeds produced on saidemasculated plants of the periwinkle line, and f) germinating saidharvested seed to produce a periwinkle plant.
 30. A first generation(F₁) hybridperiwinkle plant produced by growing said hybrid periwinkleseed of claim
 29. 31. An F₁ hybrid periwinkle plant having a totalalkaloid content of about 2.0% to about 6.5% and having a pedigree whichincludes the plant 8424-3.
 32. An F₁ hybrid periwinkle plant accordingto claim 31, wherein said total alkaloid content is between about 2.0%and about 3.0%.
 33. An F₁ hybrid periwinkle plant according to claim 31,wherein said total alkaloid content is between about 3.0% and about4.0%.
 34. An F₁ hybrid periwinkle plant according to claim 31, whereinsaid total alkaloid content is between about 4.0% and about 5.0%.
 35. AnF₁ hybrid periwinkle plant according to claim 31, wherein said totalalkaloid content is between about 5.0% and about 6.0%.
 36. An F₁ hybridperiwinkle plant according to claim 31, wherein said total alkaloidcontent is between about 6.0% and about 6.5%.
 37. Viable Catharanthusseeds and plants and succeeding generations thereof grown from the seedsdeposited under ATCC Accession No. 75636 and Catharanthus seeds andplants to which the Phytophthora resistance allele is transferred fromsaid deposited seeds or succeeding generations thereof.
 38. The methodof developing a periwinkle plant having a total alkaloid content of 2.0%or greater, comprising crossing a first periwinkle genotype with asecond periwinkle genotype, at least one of said genotypes containing atotal alkaloid content of 2.0% or greater and having a pedigree whichincludes the plant 8424-3.